L-prolyl-l-arginyl-l-volyl-l-tyrosyl-l-volyl-l-histidyl-l-prolyl-glycine

ABSTRACT

NOVEL HEPTA- AND OCTAPEPTIDES OF THE FORMULA:   2-(R1-CO-),1-(NH2-C(=NH)-NH-(CH2)3-CH(-NH-R)-CO-NH-CH(-   CH(-CH3)2)-CO-NH-CH(-CH2-(1,4-PHENYLENE)-OH)-CO-R2-NH-   CH(-CH2-C&lt;(-NH-CH=N-CH2-))-CO-)PYRROLIDINE   WHEREIN R IS HYDROGEN, SUCCINYL, L-ASPARTYL, SARCOSYL, LSERYL, SUCCINAMYL, L-PROLYL, GLYCYL, OR D- OR L-ASPARAGINYL; R1 IS AN L-ALANINE, L- OR D-LEUCINE, GLYCINE, LISOLEUCINE OR B-ALANINE RESIDUE; AND R2 IS L-VALYL, OR LALANYL UPON INTRAVENOUS INFUSION TO ANIMALS INHIBIT BLOOD PRESSURE RESPONSE TO ANGIOTENSIN AMIDE.

United States Patent "ice $75,212:

1 2 3,751,404 BOC-Asn-Arg (N0 Val Tyr(O-Bzl)-Val-His(N L-PROLYL-L-ARGINYL-L-VOLYL-L-TYROSYL-L- Bzl)-Pro-Ala-polymer(A).BOC-Alaresinester (5 g., VOLYIFL-HISTIDYL- 0.5 mMol/g.) was placed in a rocking type 200- ml.

iZ fi g P i s Merrifield reaction vessel. The resin was swelled in gi 'r gfii b g i, f ig 5 chloroform (analytical grade) by rocking for 20 min. No b a confinuationdmpart of applications and thereafter washed with three 50 ml. portions of 43,595, Jl'me 4, 1970 and sen 17,920 Man 9 I glacial acetic acid. The duration of each wash operation 1970. This application Feb. 12, 1971, Ser. No. 115,045 was The t-butoxycarbonyl protecting Int. Cl. C070 103/52 group was removed by IN HCl in anhydrous acetic acid US. Cl. 260-1125 1 Claim 10 by rocking for 40 min. The resin was successively washed three times each with acetic acid, absolute ethanol and with N,N-dimethylformamide. The resulting hydrachlo- ABSTRACT OF DISCLOSURE ride of ananyl-resinester was neutralized with 10% solu- Novel heptaand octapeptides of the formula: tion of triethylamine in dimethylformamide by rocking i H1NC|}=NH HN NH Hs0\ CH; Cg 2 H2 R-NH-JJH-C ONE-()H-C ON H-(BH-OO-Rr-NH-(EH-C ON-CHCOR1 wherein R is hydrogen, succinyl, p y sarcosyl, L- for 10 min. Thereafter the resin was washed with three ry i y -p p y y y or Or p 25 portions each of dimethylformamide, abs. ethanol, chloro- Y 1 an Or f i y form and methylenechloride and the solution of 8.5 mMol lsoleucme or B'alamne resldue; and R2 15 or (three fold excess) of BOC-proline in 40 ml. of methylalanyl upon intravenous infusion to animals inhibit blood enechloride was added A min rocking period was Pressure response to anglotensm amldeallowed to give time for the amino acid derivative to penetrate the resin. Then 8.5 mMol of N,N'-dicyclohexyl- This application is a continuation-in-part of our 00- carbodiimide in methylemchlol'ide Was pending applications Ser. No. 43,595, filed June 4, 1970, added and the coupling was allowed to proceed for 12 and Ser. No. 17,920, filed Mar. 9, 1970. hours with rocking. Thereafter the resin was washed with three portions each of methylenechloride, absolute ethanol and acetic acid and so was prepared for the next This invention relates to polypeptides. More particularly it is concerned with heptaand octapeptides of the formula: deprotection step with HCl in acetic acid as described :N HNC=NH HIL- NH H30 CH| (in... a H. H, n R-NH-JJH-C ONE-(E H-C ONH-H-CO-R -NHH-C o CHOOR wherein R is hydrogen, succinyl, L-aspartyl, sarcosyl, L- above. The Washing, neutralization and coupling steps seryl, succinamyl, L-prolyl, glycyl, or D- or L-aspraginyl; were performed by the described method using BOGHis R is an L-alanine, L- or D-leucine, glycine, L-isoleucine (N BzD-OH, BOC-Val-OH, BOC-Tyr(O-Bzl)-OH and or p-alanine residue; and R is L-valyl, or L-alanyl. BOC-Arg(NO )-OH. Change was made in the coupling The peptides of this invention possess pharmacological steps of the BOC-His (N -BzD-OH and BOC-Arg(NO activity. They are capable of inhibiting the pressor efiect OH in which cases a dimethylformamide-methylenechloof angiotensin amide upon blood pressure. Thus when ride (2:1) mixture was used as solvent. The coupling of administered by intravenous infusion to pithed rats in a Asn to the deprotected resin-heptapeptide was performed very small amount of 20 mcg./kg./rnin., the pressor elfect using BOC-Asn-ONP (BOC-Asparagine-p-nitrophenylof angiotensin amide similarly administered is inhibited. ester) in dimethylformamide by rocking the mixture for By virtue of this inhibitory property upon angiotensin 72 hours, amide induced blood pressure elevation, the peptides of After the last coupling step the resin-peptide Was this invention are valuable agents for counteracting hyperwashed with dimethylformamide, ethanol, acetic acid and tension due to angiotensin amide. They are also capable ethanol and dri d in vacuo over P 0 The weight of the of inhibiting hypertension in acute unilateral renal hyperresin-peptide was 7.3 g. tensive rats upon intravenous infusion. H-Asn-Arg(NO Val Tyr(O Bz1)-Val-His(N The heptaand octapeptides of this invention are B 1)-Pm-A1a-OH-2HBr(B).-'Ihe resin-peptide I (7.3 readily prepared in accordance with known methods for prepared a des ribed above, was suspended i 25 Preparing p p SllCh methods involve the building of ml. dry trifluoroacetic acid and a stream of dry HBr was a linear Chain of aminO acids through repetitive amide passed through at a slow rate. After 20 min. the resin linkages employing in such sequential alignment the neceswas filt d 11 and treated once more with sary protective groups susceptible of ready removal by conventional cleavage methods. The adaptation of such HBr/CF3CO0H methods to the peptides of this invention is described heremafter m exemplary fashlon by the followmg: for 40 min. The filtrates were evaporated to dryness L-Asparaginyl-L-arginyl-L-valyl-L-tyrosyl-L-valyl-L- in vacuo at 20, the oily residues were precipitated with histidyl-L-prolyl-L-alanine abs. ether and the product was dried in desiccator over potassium hydroxide. The overall yield was 1980 mg. (59.8%) of the protected octapeptidedihydrobromide.

H-Asn-Arg-Val-Tyr-Val-His-Pro Ala OH (C) .The protected peptide (B) (1.0 g.) was dissolved in 20 ml. of an acetic acid-dioxane-water (4:421, v./v.) mixture and hydrogenolysed over Pd/BaSO catalyst (0.5 g.)) for 48 hrs. at atmospheric pressure. After that time a new portion (0.2 g.) of catalyst was added and the hydrogenation was continued for 24 hours. The filtered and diluted solution was lyophilized. The yield was 750 mg. (M.P. 150165). (Calcd. for dihydrobromide: 15.32% Br: Found: 13.36% Br.)

The crude product was purified by gel permeation chro matography on Sephadex 6-25 in 0.2 M acetic acid. The fractions containing the pure peptide were combined and ]D =-s9.4 (c.=0.2, IN AcOH) 1578 -61.1 (c.=0.2, IN AcOH) Amino acid analysis: Ala: 1.00; Ar g: 1.00; Asn: 1.09;

His: 0.90; Pro: 1.04;Tyr: 0.75;Va1: 1.94.

In similar fashion other peptides of this invention were prepared by introducing the requisite unit at the appropriate stage. The peptides thus prepared are set forth in the tables herebelow:

TABLE I TLC on Merck TLC on Merck Silica Gel F-254 Cellulose F-254 plates plates Butanol-acetic acid-water (6:2:3 v./v.) Specific rotation ninhydrin detection (concentration, Peptide (formula, mol. weight) solvent) Rt Rf H-asn-Arg-Val-Tyr-Val-His-Pro-Ala-OH 2s= -81.33 0. 20 0. 50 (C43HQ6N14011, III-W. 955.1) (C.=0.22, IN AC OH) H-Asn-Arg'Val-Tyr-Val-His-Pro-Lcu-OH M 23: 70.55 0. 15 0. 65 (C HnNuOn, IILW. 997.2) (0.=1, IN ACOH) H-asn-Arg-Val-Tyr-Val-His-Pro-Leu-OH [(119 -87.32 0. 1O 0. 60 (CluHnNnOn, IJLW. 997.2) (0.=0.2, IN Ac 0H) Suceinyl-Arg-Val-Tyr-Val-His-Pro-Ala-OH 01.90 1 0. 40 1 0. 70 (CflHMNlZOiZ, IILW. 941.1) (0.=1, IN AG OH) H-Asp-Arg-Val-Tyr-Val-His-Pro-Ala-OH [a] -101.62 0. 03 0. 45 (O43H54N13012, IILW. 956.1) (c.=0.3, IN Ac OH) H-Arg-Val-Tyr-Val-His-Pro-Ala-OH [6611) -68 31 0. 02 0. 20

( m eoNnOo, m.W. 841.0)

(c.=0.24 IN' Ac on I Pauly's reagent.

TABLE II TLC on Merck Silica Gel F-254 plates (ninhydn'n detection) Butanol-acetic acid- Specification rotation Butanol-acetic acldpyridine-water Peptide (formula, mol. weight) (concentration, solvent) Water (6:2:3:v./v.) (9:2:7:6:v./v.)

H-Sar-Arg-Val-TyrHis-Pro-Ala-OH [a] 84.41 0. 05 0. (C42II05N13010, m.w. 912.11) (c=0.24, IN AcOH) H-Ser-Arg-Val-Tyr-His-Pro-Ala-OH M 69.00 0. 02 0. 38 (C42H 5NmOu, HLW. 928.11) (C.=O.2LIN AOOH H-Asn-Arg-Val-Tyr-Val-His-Pro-leu-O H [a] =54.40 0. 15 0.50 (CmHnNuou, ILLW. 997.22) (c.=0.25, IN AcOH) Succinamyl-Arg-Val-Tyr-Val-His-Pro-Ala-OH [alp 83.14 0. 10 0. 20 (C43I'Ic5N13O11, m.w. 940.12) (e.=0.25, IN AcOI-I) H-Asn-Arg-Val-Tyr-Val-His-Pro Gly-OH 2 54.90 0. 10 0. 25 (CQHMNMOH, ITLW. 04111) (0.=1.0, IN Ac OH) H-Asn-Arg-Val-Tyr-Val-His-l?r0-Ile-OH [a] 7= 33 0. 09 0.

( mHnNmOn, m.w. 997.22)

TABLE III TLC on Merck Silica Gel F-254 plates (Pauly reagent detection) Butanol-acetic-acid- Specific rotation Butanol-acetie acidwater pyridine Peptide (formula, mol. Weight) (conc., solvent) Water (622:3 v./v.) (9:2:6:7 v./v.)

H-Sar-Arg-Val-Tyr-Val-His-Pro- Gly-OH [a]n 74. 6 0. 15 0. 25 (C41H6 N13Ow, 898.10) (c.=0.57, N 1100111) H-Pro-Arg-Val-Tyr-Val-His-Pro-Gly-OH [a] 81. 0 0. 10 0. 35 (04313115133010, 924.12) (C.=0.53, N AOOH) H-Asn-Arg-Val-Tyr-Val-His-Pro-Gly-OH [a]n 67.0 0. 15 0. 32 (O HnNnOn, 941.11) (c.=0.51, N AcOH) H-Sar-Arg-Val-Tyr-Val-His-Pro- 3-A1a-OH [a] 59.0 0. 05 0. 25 C4zHu5N13010, 912.10) (0.=U.52, N AGOH) H-Asn-Arg-Val-Iyr-Val-His-Pr0-B-Ala0H [(11D22: 53.7 0. 15 O. (C-IQHGBHMOH, 955.10) (c.=0.53, N AcOH) H-Sar-Arg-Val-TyrJle-His-Pro-Leu-OH [a]r)"= 56.7 0. 15 0. 45 (CwH'nNmOm, 968.22) (0.=0.30, N AGOH) H-Asn-Arg-Val-Tyr-Ile-His-Pro-Leu-OH [a] 77.5 0. 25 0. 35

( uHnNuOn, 1011.25)

(e.=0.54, N AcOH) TABLE III-Contlnued TLC on Merck Silica Gel F-254 plates (Pauly reagent detection) Butanol-acetic-acid- Specific rotation Butanol-acetic acidwater pyridine Peptide (formula, mol. weight) (cone, solvent) water (6:223 v./v.) (922:6:7 v./v.) H-Sar-Arg-Val-Tyr-Ile-His-Pro-Ala-OH [a]n 80.5 0. 28 0. 35 (O43HMH13010, 926.14) (0-=0.54, N AGOH) H-Asn-Arg-Val-Tyr-Ile-His-Pro-Ala-OH [a]D -63.7 0. 0. 38 (CuHusNuOn, 069.17) (c.=0.52, N ACOH) H-Asn-Arg-Val-Tyr-Ala-His-Pro-A1a-OH [a]D -68.2 0. 10 0. 35 n wNmOu, 927.03) (c.=0.50, N AcOH) In the above tables, in accordance with IUPAC n0men- OTHER REFERENCES clature for distinguishing L- and D-forms of amino acids BumpuS et Biochim Biophys Acta 46 38,44 in polypeptides, L-amino acids are denoted by a first 9 1 capital letter, while D-amino acids are denoted by a first P rk t a1; Biochemistry (Wa h 6, 34584464 lower case letter. 1967).

What is claimed is: Bumpus et al.: Peptides-Chemistry and Biochemistry, 11. The compound Weinstein et al. eds., Marcel Dekker Inc., New York (1970), pp. 127-150. Elfective date: August 1968. L-prolyl-L-arginyl-L-valyl-L-tyrosy1 Schroder et al.: The Peptides, vol. II, Academic Press,

L-valyl-L-histidyl-L-prolyl-glycine New York 19 47 2 Khairallah et al.: J. Med. Chem. 13, 181-184 (1970).

References Cited 0 Khosla et al.: Biochemistry (Wash.) 1, 3417-3421 UNITED STATES PATENTS (1968)- Schwyzer et al L. i y Examiner 3,014,023 12/1961 Schwyzer et a1 260-1125 3,040,017 6/1962 Schwyzer et a1 260-112.5 US, Cl. X.R.

3,281,406 10/1966 Schwyzer et a1 260-1125 424177 

